1. Field of the Invention
The present invention provides a method and a test set for determining histamine present in samples, in particular whole blood. The method is based on a selective binding of histamine to certain materials, and is useful in the diagnosis of allergy related to specific allergens.
2. Information Disclosure Statement
It is essential for allergic patients to know which of the allergens present in their environment or diet give rise to the allergic reaction. Thus, there is a pressing need for the development of accurate, precise, easily applicable and inexpensive methods for establishing which allergens are relevent for the patient.
Methods based on imitation of the in vivo reaction involve the following:
It is known that allergenicity may be evaluated by exposing certain cells, obtained from the patient, to the allergens considered to be of importance, thus inducing a histamine release from said cells, and subsequent measuring the amount of histamine released.
A method based on this principle is described in U.S. Pat. No. 4,550,085. In its more general aspect, the patent discloses a method for detecting or determining histamine in body fluids which comprises: contacting a sample of the fluid with glass microfibers deposited onto a carrier in such quantitative proportions between the glass microfibers and the fluid as will permit the histamine amount to be detected or determined to be bound to the microfibers; and
registering or measuring the bound amount of histamine.
In particular a method for detecting histamine possibly released by allergic reaction in body fluids is disclosed wherein the body fluid is contacted with a test allergen and the possibly released histamine bound to the fibers is registered directly or competitively.
According to the working examples in the above mentioned patent the cell phase contacted with the allergens in question was a specially prepared blood cell suspension, namely a cell suspension enriched with leucocytes. To prepare this suspension, blood was drawn from the patient, and the erythrocytes were sedimented. The leucocyte-enriched plasma layer obtained was carefully transferred to another container, suspended in a buffer, and then centrifuged in order to isolate the leucocytes from the major part of the plasma. The supernatant obtained was removed, and the pelleted leucocytes were resuspended in a buffer. This step was further repeated to yield a cell suspension especially enriched with basophile leucocytes.
Accordingly, the sample contacted with the glass microfiber deposited on the carrier was not whole blood as such, but a blood sample which had been subjected to several intermediate treatments.
Pending U.S. divisional application Ser. No. 791,722 is directed to an analytical agent for use in histamine detection particularly in accordance with the method of U.S. Pat. No. 4,550,085, which comprises individual glass microfibers fixed onto a carrier, said agent adapted for use in the detection or determination of histamine possibly released by allergic reaction in body fluids which further comprises the reversible deposition of at least one test allergen.
Based on the same priority application DK 2982/81 as the U.S. Pat. No. 4,550,085, an international application WO 83/00229 was filed. The only essential difference with relation to the U.S. patent and its divisional application is the provision of a further example representing a preferred embodiment using a microtiter plate as a means for carrying out a direct histamine determination. Based on this international application, a number of patents have been granted, e.g. EP patent no. 82,862, FI patent no. 74,818 and AU patent no. 563,306.
The method according to the above patent family has given rise to a number of articles proving its reliability.
In Agents and Actions, vol. 14, 3/4 (1984), p. 414-416 the histamine test according to application WO 83/02229 is further elucidated. The test was performed on washed blood.
In order to avoid loosing of the microfibres during the various washing steps the crushed glass microfibres were fixed to the bottom of the microtiters plates with a water soluble glue.
In Allergy, vol. 42, (1987), p. 366-373 the method according to WO 83/00229 is applied to compare the microfibre-based histamine analysis with conventional histamine release assays, e.g. the skin prick test and bronchial provocation test).
The method is reported for samples of blood washed by means of Pipes-AMC buffer in order to avoid trapping of the blood cells into the microfibres.
During studies of radioenzymatic assay for histamine Verburg and Henry, Agents and Actions, vol. 14, 5/6, (1984), p. 633-636 investigated adsorption of histamine by glass surfaces in order to obtain techniques to limit the phenomenon. It was reported that potassium phosphate buffers completely prevent histamine binding, that bovine serum albumin blocked adsorption, but that Tris-buffer at pH 7.8 and K.sub.2 EDTA limited adsorption.
According to FR patent no. 1,443,167, C.A. Vol. 66 (1967), p. 4642 agglomeration of mineral fibers, i.e. rock wool, slag wool, glass wool, is known to improve mechanical characteristics and better resistance to moisture by bringing a binder, e.g. a starch paste or PhOH-HCHO-resin, on the fibers and then evaporate the solvent and polymerise the resin. Nothing is said about utilization of such agglomerates for analytical purposes.
The above mentioned prior art, in particular the patent family based on DK application no. 2982/81 is incorporated herein by reference.